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Simultaneous determination of 10 mycotoxinsin grain by ultra-high-performance liquidchromatography–tandem mass spectrometry using13C15-deoxynivalenol as internal standardP.G. Jin a , Z. Han b , Z.X. Cai a , Y.J. Wu b & Y.P. Ren aa Zhejiang Provincial Center for Disease Prevention and Control, Hangzhou, 310051, Chinab College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China

Version of record first published: 11 Oct 2010

To cite this article: P.G. Jin, Z. Han, Z.X. Cai, Y.J. Wu & Y.P. Ren (2010): Simultaneous determination of 10 mycotoxins ingrain by ultra-high-performance liquid chromatography–tandem mass spectrometry using 13C15-deoxynivalenol as internalstandard, Food Additives & Contaminants: Part A: Chemistry, Analysis, Control, Exposure & Risk Assessment, 27:12,1701-1713

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Food Additives and ContaminantsVol. 27, No. 12, December 2010, 1701–1713

Simultaneous determination of 10 mycotoxins in grain by ultra-high-performance

liquid chromatography–tandem mass spectrometry using13C15-deoxynivalenol

as internal standard

P.G. Jina, Z. Hanb, Z.X. Caia, Y.J. Wub and Y.P. Rena*

aZhejiang Provincial Center for Disease Prevention and Control, Hangzhou, 310051, China; bCollege ofPharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China

(Received 17 April 2010; final version received 17 August 2010)

An ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method forsimultaneous determination of 10 mycotoxins in grain was developed. The selected mycotoxins were:deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, nivalenol, fusarenon X, moniliformin,zearalenone, zearalanone, ochratoxin A and ochratoxin B. The samples were extracted with aqueous acetonitrile(84 : 16, v/v) and purified by reliable laboratory-made mixed cartridges. The analytes were separated on anAcquity UPLC HSS T3 column (100� 2.1mm, 1.8mm) and eluted with a mobile phase of water containing0.2% aqueous ammonia and acetonitrile/methanol (90 : 10, v/v). All mycotoxins were detected with a WatersMicromass Quattro Ultima Pt tandem quadrupole mass spectrometer operating in negative electrosprayionization using multiple reaction monitoring mode. Accurate determination was achieved by employingcommercial 13C15-deoxynivalenol as internal standard, which compensated for target loss and eliminated matrixeffects. The established method was further validated by determining the linearity (R24 0.9990), averagerecovery (75.8–106.5%), sensitivity (limit of quantitation 0.09–8.48 mg kg�1) and precision (relative standarddeviation� 6.9%). It was shown to be a suitable method for simultaneous determination of 10 mycotoxins ingrain. Finally, a total of 69 corn samples randomly collected from eastern and northern China were analyzed. Theresults showed that deoxynivalenol was the most frequently detected contaminant, whilst 3-acetyldeoxynivalenol,15-acetyldeoxynivalenol, nivalenol, zearalenone, zearalanone, fusarenon X and moniliformin also occurredfrequently. Ochratoxin A and ochratoxin B were present only in trace amounts in a small number of samples.

Keywords: method validation; LC/MS; mycotoxins; ochratoxin A; zearalenone; trichothecenes; cereals and grain

Introduction

Food contamination with toxigenic moulds has

attracted increasing attention over the last three

decades. Most grain, such as wheat, maize and rice,

can be infested by filamentous and microscopic fungi.

Some genera can produce toxic secondary metabolites,

namely mycotoxins, which impact on food safety

(Kuiper-Goodman 1995). F. graminearum, F. cul-

morum, F. crookwellense and F. poe (Bottalico 1998)

can produce type B-trichothecene mycotoxins, includ-

ing nivalenol (NIV), deoxynivalenol (DON), fusarenon

X (Fus X), 3-acetyldeoxynivalenol (3-ADON) and

15-acetyldeoxynivalenol (15-ADON). Fusarium spp.,

mainly F. graminearum, F. culmorum, F. equiseti and

F. crookwellense (Bottalico 1998), can produce zear-

alenone (ZON) and zearalanone (ZAN), which are

macrocyclic lactone derivatives of resorcinol acid.

Ochratoxin A (OTA) and ochratoxin B (OTB) are

secondary metabolites produced by several species of

Aspergillus (A. carbonarius) and Penicillium (P. verru-cosum) moulds. Moniliformin (MON) is produced byF. avenaceum and usually accumulates in significantamounts in wheat kernels. Acute and chronic ingestionof these mycotoxins by humans and animals can elicita variety of toxic effects, i.e. type B-trichotheceneslead to vomiting, diarrhea, food refusal, anemia,hemorrhage, immunosuppression and necrosis(D’Mello et al. 1999); OTA and OTB have potentialnephrotoxic, teratogenic, carcinogenic and immuno-suppressive effects. Despite the relatively low acutetoxicity of ZON, it exhibits distinct estrogenic andanabolic properties in several animal species. Becausegrains are primarily used for food, strict regulatorylimits for the amounts of the mycotoxins in grains havebeen established. In the European Union, the maxi-mum contents were established at the following levels:1250 mg kg�1 for DON, 100 mg kg�1 for ZON and5 mg kg�1 for OTA in unprocessed cereals (EuropeanCommission 2002, 2005, 2007).

*Corresponding author. Email: renyiping@263.net

ISSN 0265–203X print/ISSN 1464–5122 online

� 2010 Taylor & Francis

DOI: 10.1080/19440049.2010.517222

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Various techniques have been used for the extrac-tion of mycotoxins from corn matrices and for thesubsequent purification of the extracts. Solid-phaseextraction (SPE) is the most often applied clean-upmethod. The main materials of SPE were silica gel,florisil and ion-exchange resin as reported previously(Cohen and Lapointe 1982; Mateo et al. 2002).Recently, Mycosep columns have become more fre-quently used (Razzazi-Fazeli et al. 2002; Castillo et al.2008). The application of immunoaffinity columns hasalso been successful for the concentration and purifi-cation of several mycotoxins (Bascaran et al. 2007;Kabak 2009). However, there is no clean-up methodapplicable for simultaneously dealing with all of the10 mycotoxins. For quantitation, some analyticaltechniques, such as thin-layer chromatography (TLC)(Eppley et al. 1986; Solovey et al. 1999; Omurtag andYazicioglu 2001), gas chromatography (GC)(Schollenberger et al. 1998), capillary electrophoresis–laser induced fluorescence (CE–LIF) (Maragos andAppell 2007), high performance liquid chromatogra-phy (HPLC) and enzyme-linked immunosorbent assay(ELISA) (Gilbert et al. 1992; Grabarkiewicz-Szczesnaet al. 2001; Krska and Josephs 2001; Krska et al. 2001),have been developed. Poor separation, unsatisfiedaccuracy and low sensitivity limit TLC application.Although ELISA is a very sensitive and rapid method,it may cause false positive results. Derivatization isneeded in HPLC combined fluorescence detection,which is time-consuming (Gonzalez-Osnaya et al.2008). HPLC–MS/MS, which consumes smallamount of sample and solvent, is a robust methodfor determining mycotoxins (Razzazi-Fazeli et al. 2002;Berthiller et al. 2005; Klotzel et al. 2005, 2006; Sulyoket al. 2006; Spanjer et al. 2008; Di Mavungu et al.2009); however, molecules originating from the samplematrix that co-elute with the analytes can interfere withthe ionization process in the mass spectrometer,causing ionization suppression/enhancement (Kebarleand Tang 1993; Bogialli et al. 2003). The problemmight be overcome by two approaches: one is to useexternal matrix calibration; the other is to add internalstandards that behave similar to the analytes (Haublet al. 2006). In the present study, the sample pretreat-ment, chromatographic conditions and the internalstandard have been optimized to minimize matrixeffects.

The aim of this study is to develop a reliable ultra-high-performance liquid chromatography–tandemmass spectrometry (UHPLC–MS/MS) method using13C15-DON as internal standard for simultaneousdetermination of 10 mycotoxins commonly found ingrains. For this purpose, different SPE materials weretested for their suitability for UHPLC–MS/MS anal-ysis and a simultaneous clean-up method for allmycotoxins was developed. The validation details,including recovery, sensitivity, accuracy and

repeatability, are presented and evaluated. Finally,

the established method was successfully applied to

analyze 69 corn samples randomly collected from

northern and eastern China.

Materials and methods

Chemicals and reagents

The standards for DON, 3-ADON, Fus X, 15-ADON,

NIV, MON, ZAN, ZON, OTA, OTB and 13C15-DON

(IS) were purchased from Alexisa (San Diego, CA,USA). The chemical structures of the 10 mycotoxins

in the present study are shown in Figure 1.Acetonitrile and methanol were of HPLC grade

(Merck, Darmstadt, Germany). Water was purified

using a Milli-Q Gradient A 10 system (Millipore,

Billerica, MA, USA). Aqueous ammonia and other

solvents were of analytical grade.

Apparatus

UHPLC analyses were performed using a Waters

Acquity Ultra High Performance LC system (Waters,

Milford, MA, USA). Separation was achieved on an

Acquity UPLC HSS T3 column (100� 2.1mm, 1.8 mm;Waters) with a mobile phase flow-rate of 0.25ml

min�1. The mobile phase consisted of (A) water

containing 0.2% aqueous ammonia and (B) acetoni-

trile/methanol (90 : 10, v/v). A gradient elution pro-

gram was applied as follows: 0–1min hold on 2% B,

1–4min linearly increased from 2 to 10% B, 4–4.7min

linearly increased from 10 to 14% B, 4.7–6min hold on

14% B, 6–6.2min linearly increased from14 to 24% B,

6.2–11min hold on 24% B, 11–11.3min linearly

increased to 100% B, hold for another 0.5min thendecreased to 2% B. A subsequent re-equilibration time

(2min) should be performed before the next injection.

The injection volume was 5 ml.Determination was performed using a Waters

Micromass Quattro Ultima Pt tandem quadrupole

mass spectrometer (Micromass, Manchester, UK) with

the following settings: capillary voltage of 3.0 kV,

desolvation temperature of 350�C and source block

temperature of 120�C. The cone and desolvation gas

flow were 60 and 570 l h�1, respectively. Quantitation

was performed in multiple reaction monitoring

(MRM) mode and the optimization for each myco-

toxin was performed during infusion. The parameters

and collision energies of precursor ions and product

ions selected for the analysis of the mycotoxins in ESI�

mode are shown in Table 1. Data acquisition and

processing were performed using MassLynx v4.1.

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Preparation of standard solutions

Accurately weighed solid portions of each mycotoxin

standard and the internal standard were dissolved inacetonitrile to prepare 0.1mgml�1 stock solutions.

A mixed stock solution, containing 1 mgml�1 for theOTA and OTB, 50 mgml�1for MON and 10 mgml�1

for NIV, DON, 3-ADON, 15-ADON, Fus X, ZAN,

ZON, was prepared in acetonitrile and stored in the

dark at 4�C for no more than 1 month. Working

standard solutions were prepared from these stock

solutions and were diluted step by step with the blank

matrix to prepare a sequence of working solutions,

while the IS was directly diluted to 254 ngml�1

immediately before use.

Figure 1. Chemical structures of the 10 mycotoxins.

Table 1. Parameters and collision energies of precursor and product ions, concentration range (mg kg�1), limit of detection(LOD)b, limit of quantitation (LOQ)c and linearity (R2) for various mycotoxins.

NamesPrecursorion (m/z)

Primary production (m/z)

Collisionenergy (eV)

Secondary production (m/z)

Collisionenergy (eV)

R2a Range(mg kg�1)

OTA 401.8(-H) 357.8 18 210.8 26 0.9992 0.2–50OTB 367.9(-H) 323.9 16 279.9 24 0.9991 0.2–50NIV 311.0(-H) 280.9 12 138.0 14 0.9992 2–500ZON 317.0(-H) 174.9 22 273.0 18 0.9995 2–500DON 294.9(-H) 264.9 10 138.1 12 0.9992 2–500MON 97.0(-Na) 41.4 24 0.9999 10–50003-ADON 337.0(-H) 307.0 10 217.0 16 0.9992 2–50015-ADON 337.0(-H) 150.0 8 276.9 14 0.9995 2–500ZAN 319.0(-H) 275.0 18 205.0 20 0.9992 2–500FUS X 352.9(-H) 262.9 10 187.0 18 0.9996 2–50013C15-DON 310.1(-H) 279.1 10 261.1 12

Notes: aR2: regression coefficient.bLimit of detection (S/N¼ 3).cLimit of quantitation (S/N¼ 10).

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Preparation of laboratory-made mixed cartridges

A 1.6 g sample of silica gel was accurately weighed intoa 6ml hollow solid-phase extraction cartridge and thecartridge was shaken to compact the silica gel. Then,florisil (0.45 g) and kieselguhr (0.45 g) were added onthe silica gel. Finally, a cribriform plate was put onthe top layer to ensure the top surface was smoothand flat.

Samples

A total of 63 samples were collected from local storesin six cities (Shaoxing (9), Hangzhou (9), Jiaxing (11),Ningbo (11), Jinhua (14), Quzhou (9)) in Zhejiangprovince of eastern China. These areas are warm,humid and rainy. The other six samples were from Jilinprovince in northern China, where the weather is dryand cold.

Sample preparation

Portions (2.5 g) of corn were spiked with 50.8 ng ofI.S. by adding 200 ml of a 254 ngml�1 solution. Thesamples were ultrasonicated for 45min with 10mlacetonitrile/water (84 : 16, v/v). The mixture was fil-tered and a 5ml aliquot of filtrate purified by thelaboratory-made mixed cartridges and eluted with 3mlmethanol. The effluents, including the loading solutionand the eluent, were combined and dried by nitrogen at40�C. The residue was dissolved by 1ml combinedsolution (acetonitrile/10mmol l�1 ammonium acetate,10 : 90, v/v).

Results and discussion

Optimization of UHPLC conditions

Selection of UHPLC columns

In a previous study, ZON, ZAN and 3-ADON, 15-ADON could not be separated (Ren et al. 2007). To geta complete separation of the 10 mycotoxins, selectionof UHPLC–MS/MS columns with high separationefficiency is important. Several columns with differentparticle sizes and lengths were tested and two columns,i.e. (1) Acquity UPLC BEH C18 column (100�2.1mm, 1.7mm), (2) Acquity UPLC HSS T3 column(100� 2.1mm, 1.8mm), were selected as the final can-didates. Symmetrical and sharp peaks were got on bothcolumns and the sensitivities were almost the same.However, all 10 mycotoxins could be completelyseparated when choosing column 2, while 3-ADONand 15-ADON could not be separated on column 1.Therefore, Acquity UPLC HSS T3 column(2.1� 100mm, 1.8mm particle size) was selected asthe analytical column.

Selection of mobile phase

Different mobile phases were tested in the work: (1)methanol/water, (2) acetonitrile/water, (3) acetonitrile/water (containing 10mM ammonium acetate), (4)acetonitrile/water (0.2% aqueous ammonia), (5) ace-tonitrile/water (containing 0.15% diethylamine,adjusted to pH 3.0 with acetic acid) (Figure 2).It was clear that the responses of the 10 mycotoxinswere greatly improved and higher sensitivity wassubsequently obtained when No. 4 mobile phase wasselected compared to other mobile phases, due to theESI� ionization efficiency. Under such situation, nicepeak shapes were also achieved; however, 3-ADONand 15-ADON could not be completely separated. Forthis purpose, ratios of acetonitrile/methanol, varyingfrom 90 : 10 to 50 : 50, were also tested. Results ofmultiple injections showed that all mycotoxins couldget a complete separation when acetonitrile/methanol(90 : 10, v/v) was selected. Based on these consider-ations, (A) water containing 0.2% aqueous ammoniaand (B) acetonitrile/methanol (90 : 10, v/v) was selectedas the mobile phase in the present study.

Selection of column temperature

Column temperature was also tested to get a betterseparation. The results showed that, as the columntemperature increased in the range 25–45�C, theretention time decreased more or less. All mycotoxinscould be completely separated with a column temper-ature of 40�C.

Optimization of sample pretreatment

No commercial cartridge was suitable for simultaneousdetermination of 10 mycotoxins. In a previous study(Ren et al. 2007), a Mycosep 226 Multifunctionalcartridge was used, but the recoveries of OTB andMON were very low. In this study, we wished to finda more suitable clean-up method for the simultaneousdetermination of all mycotoxins.

Four SPE materials (silica gel, florisil, aluminiumoxide and kieselguhr) were tested for their suitabilityfor clean-up of sample extracts. Firstly, we evaluatedthe recovery performance of all materials by passing amixed standard solution (10 ngml�1 for OTA andOTB, 500 ngml�1 for MON and 100 ngml�1 for NIV,DON, 3-ADON, 15-ADON, Fus X, ZAN, ZON)through the columns filled with one material. Allmaterials, except aluminium oxide, showed satisfactoryrecoveries in the range 74–108%. Then, the recoveriesof mycotoxin-spiked corn extracts in each adsorbentmaterial were compared. The standards were addedinto mycotoxin-free corn extracts to obtain a workingsolution with concentrations of 10 ngml�1 for OTAand OTB, 500 ngml�1 for MON and 100 ngml�1 for

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NIV, DON, 3-ADON, 15-ADON, Fus X, ZAN, ZON.The spiked extracts were passed through thecolumns and recoveries were in the range 61.8–89.3%. In the next step, purification efficiency wastested. The spiked corn extract was cleaned-up with thecolumns filled with different materials at differentratios and the sensitivity compared. Based on these,a reliable laboratory-made mixed cartridge wasdeveloped.

In this study, the sensitivities of the 10 mycotoxinsin spiked corn extracts, cleaned-up with the lab-mademixed cartridges or not were also compared. It wasshown that sensitivities were significantly improvedwhen the spiked extracts were purified with the mixedcartridges (Table 2). As the proposed cartridgesshowed satisfactory recoveries and purification effi-ciencies for the crude extracts, the laboratory-mademixed cartridges were shown to be suitable for sampleclean-up.

Ascertaining the internal standard

Generally speaking, only a substance with similarmolecular structure and physicochemical property

could meet the requirements of an internal standard.

In a previous study (Ren et al. 2007), ZAN was chosen

as the I.S.; however, Cavaliere et al. (2005) found ZANexisted in natural samples. In the present study, ZAN

was also detected in corn samples; therefore, the I.S.should be reselected.

Isotope dilution is an accurate and effective way to

ensure analytical results. Because isotope internal

standards of the relative mycotoxins are very difficult

to obtain as commercial products, there are only a few

reports on the determination of mycotoxins using I.S.

based on stable isotope-labeled analytes (Haubl et al.

2006; Cunha et al. 2009). Fortunately, 13C15-DON was

obtained as a commercial product in our laboratory.

The advantage was that it behaved similarly to most of

the targets during sample preparation and in the ion

source interface, and it can be separated and distin-

guished in the mass spectrometer from their different

molecular weights. To check whether the I.S. contained12C15-DON, the I.S. solution (254 ngml�1) was ana-

lyzed by UHPLC–MS/MS. The results showed that no

interference existed in both channels of the primary or

secondary daughter ions of DON, verifying the suit-

ability of the solution as an I.S.

Figure 2. Comparison of the separation and ionization efficiencies of the 10 mycotoxins among five candidate mobile phases.Peaks: 1¼MON, 2¼NIV, 3¼OTB, 4¼OTA, 5¼DON, 6¼Fus X; 7¼ZON, 8¼ZAN, 9¼ 15-ADON, 10¼ 3-ADON.

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Method validation

Linearity

Calibration curves were constructed with concentra-tion sequences of 2, 5, 8, 10, 20, 50, 80, 100, 200,500 ngml�1 for DON, 3-ADON, 15-ADON, ZAN,ZON, 0.2, 0.5, 0.8, 1, 2, 5, 8, 10, 20, 50 ngml�1 forOTA and OTB and 10, 20,50, 80, 100, 200, 500, 800,1000, 2000, 5000 ngml�1 for MON, and 25.4 ngml�1

of [13C15] -DON was included. The calibration curvesusing the isotope dilution method were created afterinjection (5ml) of each standard solution. All solutionswere prepared in blank matrix. Calibration curves werelinear with correlation coefficients (R2)4 0.9990 for allanalytes (Table 1).

Limit of determination (LOD) and limit ofquantitation (LOQ)

The detection (quantitation) limits were determined bysuccessive analyses of chromatographic extracts ofcorn spiked samples with decreasing amounts of everymycotoxin standard until a signal-to-noise ratio of 3 : 1(10 : 1) was reached. These values showed that theLOD and LOQ of the method met the legislativerequirements set by the EU for the mycotoxins(Table 2).

Recovery and precision

Recoveries were performed by the method of standardaddition. Standards were added to analyte-free cornsamples at high, intermediate and low levels (50, 5 and0.5 mg kg�1 for OTA and OTB; 5000, 500 and50 mg kg�1 for MON; 500, 50 and 5 mg kg�1 forDON, 15-ADON, 3-ADON, ZAN, ZON, NIV, FusX). The samples were pretreated as described above.If one depicts the peak areas obtained in a blankmatrix as A for standard and B for I.S., the

corresponding peak areas for standards spiked beforeextraction as A1 for standard and B1 for I.S., therelative recovery (RR) and absolute recovery (AR) canbe calculated as follows:

RR ¼A1=B1

A=B, ð1Þ

AR ¼A1

A: ð2Þ

The results of the relative recoveries and absoluterecoveries are summarized in Table 3. Compared toabsolute recoveries, relative recoveries were muchhigher, indicating that the use of 13C15-DON in thepresent study had the advantage that an accuratequantitation for all mycotoxins was achieved.

Repeatability, intra-day precision and inter-dayprecision for each compound at low, intermediateand high concentration levels were also performed.The RSDs of the intra-day precision were in the range0.5–5.5% and the RSDs of inter-day precision werein the range 1.4–6.8%. All these data revealed that theestablished method had an acceptable precision.

Applicability

Different blank cereals, including rice, buckwheat,broomcorn, bean, mung bean, glutinous rice, milletand fragrant rice, were spiked with the 10 mycotoxinstandards (50mg kg�1 for ZAN, ZON, DON, 15-ADON, 3-ADON, NIV; 500 mg kg�1 for MON;5 mg kg�1 for OTA, OTB). Then, the spiked sampleswere pretreated as described about and determined byUHPLC–MS/MS using 13C15-DON as I.S. All recov-eries were in the range 71.6–117.9% (Table 4),indicating the developed method could be used forquantitation of 10 mycotoxins in all these cereals.

Method application

Following the optimization and validation of theanalytical method, it was applied to determine con-tamination levels in corn samples for human consump-tion obtained from different areas of China. MRMchromatograms of the 10 mycotoxins in standardsolutions (a) and in contaminated samples (b) areshown in Figure 3.

The mycotoxin demonstrated great variability intype and relative proportions in the 65 contaminatedsamples (Table 5). A total of 61 samples containedDON, the most prevalent contaminant, in the range2.7–311.2mg kg�1. A total of 19 samples contained typeB-trichothecene mycotoxins, including DON, 15-ADON, 3-ADON, NIV and Fus X, with an averagedistribution of 81.6, 11.8, 1.8, 3.7 and 1.1%, respec-tively, in the range 8.8–373.1 mg kg�1. Although it was

Table 2. Comparison of sensitivities of mycotoxins cleaned-up or not with the laboratory-made mixed cartridges.

Names

Purified samples Unpurified samples

LOQ(mg kg�1)

LOD(mg kg�1)

LOQ(mg kg�1)

LOD(mg kg�1)

OTA 0.09 0.03 0.51 0.17OTB 0.12 0.04 0.61 0.20MON 8.48 2.83 18.96 6.32Fus X 1.78 0.59 4.14 1.383-ADON 0.32 0.11 1.06 0.3515-ADON 0.44 0.15 1.08 0.54ZAN 1.73 0.58 2.89 0.96ZON 1.44 0.48 5.16 1.72DON 0.48 0.16 2.08 0.69NIV 0.74 0.25 1.58 0.53

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Table

3.Relativeandabsolute

recoverytestsin

corn

samplesofUHPLC–MS/M

Smethod(n¼3).

Relativerecovery

Absolute

recovery

Highlevela(%

)X��SD

RSD%

Interm

ediate

levelb(%

)X��SD

RSD%

Low

levelc(%

)X��SD

RSD%

Highlevela(%

)X��SD

RSD%

Interm

ediate

levelb(%

)X��SD

RSD%

Low

levelc(%

)X��SD

RSD%

DON

98.9�2.9

2.9

102.5�2.3

2.2

102.2�8.2

8.0

79.6�3.0

3.8

81.6�1.4

1.7

82.2�2.7

3.3

3-A

DON

103.6�8.4

8.1

106.5�3.3

3.1

105.1�2.8

2.6

81.8�2.2

2.7

84.4�2.6

3.1

83.9�4.7

5.6

15-A

DO

98.8�5.1

5.2

97.7�4.6

4.7

100.3�6.6

6.6

77.0�3.0

3.9

79.7�1.6

2.0

81.9�4.9

5.9

NIV

93.1�4.0

4.3

95.8�4.0

4.2

92.9�4.1

4.4

79.0�1.6

2.0

82.5�1.7

2.1

83.9�3.4

4.1

FX

100.7�1.9

1.9

102.4�5.1

5.0

104.6�4.3

4.1

80.0�1.7

2.1

88.4�2.0

2.3

86.9�3.9

4.5

ZON

77.9�3.6

4.6

78.4�3.5

4.5

78.1�4.1

5.2

63.5�2.4

3.8

63.2�2.8

4.4

67.3�2.4

3.6

ZAN

78.3�2.4

3.1

75.8�1.3

1.7

76.2�5.9

7.7

64.4�2.4

3.7

64.8�3.9

6.0

66.9�1.9

2.8

OTA

95.5�9.2

9.6

99.1�3.7

3.8

98.7�6.5

6.6

80.5�4.4

5.5

82.8�3.2

3.9

83.3�4.1

4.9

OTB

92.8�4.4

4.7

95.4�3.5

3.7

90.1�6.2

6.9

77.9�1.3

1.7

81.5�1.9

2.3

80.1�4.5

5.6

MON

85.3�4.9

5.8

86.5�4.4

5.1

87.6�4.5

5.2

69.5�4.2

6.0

70.5�1.9

2.7

72.4�2.5

3.5

Notes:

a50mg

kg�1forOTA

andOTB;5000mg

kg�1forMON;500mg

kg�1forDON,15-A

DON,3-A

DON,ZAN,ZON,NIV

,FUSX.

b5mg

kg�1forOTA

andOTB;500mg

kg�1forMON;50mg

kg�1forDON,15-A

DON,3-A

DON,ZAN,ZON,NIV

,FUSX.

c0.5mg

kg�1forOTA

andOTB;50mg

kg�1forMON;5mg

kg�1forDON,15-A

DON,3-A

DON,ZAN,ZON,NIV

,FUSX.

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within the acceptable limits in European Union or inChina (Chinese Commission 2005; EuropeanCommission 2005, 2007), the potential risks wereassociated with the accumulative effects. A total of62 samples contained ZON ranging from 2.7 to167.2mg kg�1 and eight of them exceeded the limitsof the European Union. A total of 17 samplescontained both ZON and ZAN, with an averagedistribution of 92.6 and 7.4%, respectively, in therange 6.8–167.2 mg kg�1. This was similar to the valuereported for Italy (8–969 mg kg�1) (Cavaliere et al.2005), but a little higher than that reported from theState of Tlaxcala (3.25–83.63 mg kg�1) (Briones-Reyeset al. 2007). A total of 22 samples were contaminatedwith MON, ranging 9.2–251.5mg kg�1. A total of 13samples contained DON, 3-ADON, 15-ADON, NIV,Fus X, ZON, ZAN and MON, with an averagedistribution of 50.9, 7.3, 1.2, 2.3, 0.7, 15.3, 1.2 and21.1%, respectively, in the range 16.6–578.1 mg kg�1.Only traces of OTB in three corn samples weredetected and OTA was not detectable in all but oneof the analyzed corn samples.

Moreover, the environmental factors also affect thetype of mycotoxin produced and accumulated.Fusarium strains are suitable for producing highcontents of DON in temperate regions (Bottalico1998; Mubatanhema et al. 1999; Cavaliere et al.2005). In Zhejiang Province, the survey clearly dem-onstrated that all mycotoxins were regularly presentin cereal-based food and feed throughout all

commodities. Among 63 samples, 62 were contami-nated with mycotoxins, ranging in levels up to578.1 mg kg�1 (Table 5). Among the 10 mycotoxins,DON, 15-ADON and ZON were the major detectedcontaminants: the mean levels (occurrence) in positivesamples were 80.8 (88.7%) mg kg�1, 10.4 (88.7%)mg kg�1 and 29.6 (90.3%) mg kg�1, respectively.3-ADON, NIV, MON, ZAN and Fus X also fre-quently existed in samples, the mean levels (occur-rence) in positive samples were 2.0 (51.6%) mg kg�1, 3.1(56.5%) mg kg�1, 45.4 (34.9%) mg kg�1, 2.1 (27.4%)mg kg�1 and 0.9 (24.2%) mg kg�1, respectively. On theother hand, in Zhejiang province (eastern China), allmycotoxins showed lower levels than from Jilin prov-ince (northern China). Although the number of sam-ples did not show significant correlations betweenclimate and mycotoxins content, it was clear thathumidity and warm weather are suitable for mycotoxingrowth.

Conclusions

A reliable UHPLC–MS/MS method was developedfor simultaneous determination of 10 mycotoxins ingrain. Samples were extracted with acetonitrile/water(84 : 16, v/v) and the extracts were reconstituted by asimple and time-saving clean-up method based ona reliable and cost-efficient laboratory-made cartridge.After optimization of the chromatographic conditions,

Table 4. Average recovery tests in multiple cereal samples of UHPLC–MS/MS method (n¼ 3).

Rice (%)X�

� SD RSD%Fragrant rice (%)X�

� SD RSD%Broomcorn (%)X�

� SD RSD%Buckwheat (%)X�

� SD RSD%

DON 99.5� 4.3 4.3 98.5� 3.2 3.2 99.3� 4.2 4.2 97.6� 4.5 4.63-ADON 111.8� 3.6 3.2 113.8� 1.2 1.1 114.6� 2.4 2.0 107.3� 2.1 2.015-ADON 93.8� 2.8 3.0 104.2� 3.1 3.0 112.1� 1.5 1.3 105.5� 4.3 4.1NIV 91.7� 1.5 1.6 99.5� 2.7 2.7 104.1� 2.7 2.6 93.3� 1.3 1.4FX 103.4� 1.6 1.6 102.1� 3.2 3.1 104.8� 2.1 2.0 106.5� 3.3 3.1ZON 80.2� 3.1 3.9 85.0� 1.9 2.1 87.5� 1.6 1.8 84.5� 4.0 4.7ZAN 79.6� 2.9 3.6 81.2� 3.9 4.8 83.6� 2.3 2.8 82.9� 2.9 3.5OTA 87.7� 6.1 6.9 83.1� 2.8 3.3 78.5� 3.2 4.1 84.1� 3.7 4.4OTB 103.8� 5.7 5.5 100.9� 4.7 4.7 104.9� 4.5 4.3 104.4� 2.5 2.4MON 102� 1.4 1.4 105� 3.8 3.6 82.5� 4.3 5.2 86.4� 3.1 3.6

Mung bean (%)X�

� SD RSD%Bean (%)

X�

� SD RSD%Glutinous rice (%)X�

� SD RSD%Millet (%)

X�

� SD RSD%

DON 101.2� 3.8 3.8 97.5� 2.1 2.2 95.8� 2.8 2.9 96.8� 4.1 4.23-ADON 117.9� 2.7 2.3 108.8� 3.0 2.8 95.9� 2.4 2.5 103.1� 2.8 2.815-ADON 102.8� 3.6 3.5 110.8� 2.6 2.4 89.0� 2.6 2.8 96.8� 3.1 3.2NIV 97.3� 2.8 2.9 101.7� 1.8 1.8 102.1� 3.5 3.4 100.9� 3.3 3.3FX 107.8� 2.2 2.0 108.1� 4.0 3.7 104.9� 2.5 2.4 102.4� 5.7 5.6ZON 94.0� 6.1 6.5 80.8� 3.1 3.9 71.6� 2.5 3.5 76.9� 4.8 6.3ZAN 90.8� 5.5 6.1 75.3� 2.8 3.7 73.2� 4.3 5.9 72.9� 5.3 7.3OTA 96.6� 4.8 5.0 88.4� 2.0 2.2 87.4� 3.9 4.5 82.8� 4.3 5.2OTB 95.2� 2.8 2.9 91.5� 3.5 3.8 94.6� 2.9 3.1 91.3� 3.3 3.6MON 98.5� 3.4 3.5 83.1� 3.9 4.7 106� 4.1 3.9 95.2� 5.1 5.4

1708 P.G. Jin et al.

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Figure 3. MRM chromatograms of 10 mycotoxins in the standard solutions (a) and in contaminated samples (b). The mobilephase consisted of (A) water containing 0.2% aqueous ammonia and (B) acetonitrile/methanol (90 : 10, v/v) with a gradientelution program applied. Experimental conditions: Acquity UPLC HSS T3 column (100� 2.1mm, 1.8mm), the concentrationswere 10 ngml�1 for the OTA and OTB, 500 ngml�1 for MON and 100 ngml�1 for NIV, DON, 3-ADON, 15-ADON, Fus X,ZAN, ZON.

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Figure 3. Continued.

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Table

5.Incidences,meansofpositives

andconcentrationranges

(mgkg�1)oftarget

mycotoxinsin

corn

samplesrandomly

collectedfrom

differentareasofChina.

EasternChina(Zhejiangprovince)

NorthernChina(Jilin

province)

CentralZhejianga

SouthernZhejiangb

NorthernZhejiangc

Names

Incidence

(postot�

1)

Mean

(mgkg�1)

Range

(mgkg�1)

Incidence

(postot�

1)

Mean

(mgkg�1)

Range

(mgkg�1)

Incidence

(postot�

1)

Mean

(mgkg�1)

Range

(mgkg�1)

Incidence

(postot�

1)

Mean

(mgkg�1)

Range

(mgkg�1)

DON

26/29

119.8

7.7–311.2

18/23

55.2

6.4–190.4

11/11

81.7

3.6–192.8

6/6

39.4

2.7–91.2

3-A

DON

15/29

2.9

2–6.1

10/23

4.9

2.1–8

7/11

4.1

2.2–8.1

3/6

3.8

2.8–5

15-A

DON

26/29

12.8

2.6–32.5

19/23

10.0

2.1–21.4

10/11

12.3

2.8–27.8

5/6

8.1

3.1–19.9

NIV

15/29

6.0

2.1–15.3

12/23

5.0

2.5–13.8

8/11

5.5

2.5–8.9

1/6

3.2

3.2

FUSX

9/29

3.8

2.1–8.2

1/23

2.0

2.0

5/11

3.6

2.4–4.6

4/6

4.3

3–6.5

ZON

26/29

36.2

2.7–167.2

19/23

21.2

4.5–93.6

11/11

44.7

3.1–96.3

6/6

16.2

6.1–36.6

ZAN

11/29

9.8

4.1–58.6

5/23

4.1

3.1–4.9

1/11

4.2

4.2

0/6

––

OTA

1/29

0.1

0.1

0/23

––

0/11

––

0/6

––

OTB

1/29

0.5

0.5

2/23

0.45

0.4–0.5

0/11

––

0/6

––

MON

8/29

135.4

48.7–248

6/23

145.3

9.2–251.5

8/11

107.6

36.1–221.4

0/6

––

Notes:

aHangzhoucity,Shaoxingcity

andNingbocity.

bQuzhoucity

andJinhuacity.

cJiaxingcity.

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all analytes could be completely separated in less than11min, providing narrow peaks with good symmetry.After validation by determining the linearity, sensitiv-ity, recovery and precision, the established UHPLC-MS/MS method was proven to be a quick, efficient,selective and reliable method for the determination ofthe 10 mycotoxins. Finally, the established method wassuccessfully employed to analyze a total of 69 cornsamples randomly collected from eastern and northernChina.

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